Abstract Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and essential for male fertility. In rodents, SSCs can be frozen, thawed, cultured and transplanted to regenerate complete spermatogenesis and fertility. These well-established and robust methods have been used to investigate the molecular mechanisms that regulate stem cell self-renewal and differentiation; discover genes that are essential for SSC function; produce genetically modified animals and preserve valuable germlines. The same methods may have application for treating some cases of male infertility. However, translation to the human clinic is hampered because our current knowledge of stem cells and spermatogenic lineage development in humans is limited, we have few experimental tools for studying human SSCs and human SSC culture is not well established. The overarching objective of this application is to establish human SSC culture to facilitate fundamental investigation and practical application in the fertility clinic. Our multidisciplinary team has unparalleled access to human testis samples (207 samples archived to date with accrual ongoing). Prepubertal patients travel from around the world to cryopreserve their testicular tissues at the Fertility Preservation Program in Pittsburgh (directed by K. Orwig, PI) before initiating chemotherapy or radiation treatments that could cause permanent infertility. The challenge is that biopsies obtained from the testes of prepubertal patients are small and likely to contain a small number of SSCs, which may limit the regenerative potential of the cryopreserved samples. Human SSC culture may provide a solution to expand patient SSCs sufficiently to achieve robust spermatogenesis and restore fertility in survivors. Our team will use the Drop-seq microfluidics platform to discover the molecular signature of human undifferentiated spermatogonia and the surrounding somatic cells; derive extracellular matrix (ECM) hydrogels and matrix bound nanovesicles (MBVs) from porcine and human tissues; and establish serum free, feeder free conditions to maintain and expand human SSCs in culture. To demonstrate that our human SSC culture conditions are robust, we will confirm that they can be replicated between our project sites at the University of Michigan, the University of Pittsburgh and Kyoto University in Japan. Human SSC cultures will facilitate fundamental investigations in a species that is not amenable to transgenic or transplantation approaches and may enable stem cell therapies for male infertility.